The long term goal of this research is to increase understanding of the physiological and pharmacological properties of neurotransmission in the peripheral nervous system. To provide a simplified system in which to test the role of several putative transmitter substances, enteric neurons and smooth muscle cells are dissociated from the plexuses and smooth muscle layers of small intestines of neonatal rats and grown in cell cultures. The nerve and smooth muscle cells survive for up to 60 days and develop many properties similar to those of their in vivo counterparts. Different subpopulations of neurons contain immunoreactive vasoactive intestinal peptide (VIP), Substance P (SP), serotonin (5HT), somatostatin and met-enkephalin. Neurons in the cultures are excited by VIP, SP, 5HT, and acetylcholine (ACh) and inhibited by met-enkephalin. The smooth muscle retains contractility and is excited by ACh and SP and is relaxed by VIP. To facilitate finding pairs of synaptically connected cells or single neurons that innervate smooth muscle, pairs of neurons or single neurons are grown in microcultures. Intracellular recordings from pairs of neurons reveal fast excitatory cholinergic connections between many of them. In addition, 5-10% of cholinergic neurons release a second non-cholinergic transmitter that causes a slow depolarization of the target neuron. The specific aims of this proposal are: 1) To use voltage clamp and single channel recording techniques to analyze the mechanisms by which the aforementioned transmitter candidates excite or inhibit the cultured nerve and muscle cells; 2) To use voltage clamp and single channel techniques to analyze the mechanisms of synaptic potentials evoked by stimulation of single cells; 3) To compare the actions of exogenously applied transmitter candidates to those of synaptically released transmitters with respect to voltage dependence, ionic dependence, pharmacological properties and kinetics; and 4) To use immunohistochemical methods to test individual physiologically characterized neurons for their content of the transmitter candidates predicted by the physiological experiments. The major health-relatedness of this work is that better understanding of normal physiological processes is necessary in order to understand the ways in which these processes go awry during disease. Such understanding may in turn lead to the design, testing and development of better and more specific treatments for disease-caused perturbations of neurotransmission.